Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT- like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid- derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.